This invention generally relates to polymeric matrices for combined or individual chromatographic or electrophoretic separations of mixtures, and is in particular a mosaic matrix formed from a combination of hydrophilic or hydrophobic materials uniformly dispersed throughout a crosslinked polymer network.
Separations of biological or synthetic (e.g., pharmaceutical products, organic or inorganic mixtures) materials rely primarily on differences in molecular weight or differences in overall charge of the molecules. These separations commonly fall into two categories, chromatographic separations and electrophoretic separations.
Chromatographic separations usually involve the use of a polymeric packing or coated silica particles in combination with an elution buffer that forces the material to be separated through the matrix, where molecules move at a different rate depending on their affinity toward the packing. In case of polymer packing, the matrix may be formed of a crosslinked polymeric gel or small particles of crosslinked polymer. The porosity of the gel and/or particles effects the separation based on the molecular weight. The polymer may also be charged and separation effected by altering pH and/or ionic concentration, either by batch elutions or by gradient elution. Other specific interactions may also play a role, such as in affinity chromatography, chiral separation, ion-exchange and reverse-phase liquid chromatography.
Materials for use in chromatographic separations have been commercially available for many years, and include agarose and acrylamide gel and cellulose derivative beads such as Sephadex.TM. and Sepharose.TM., manufactured by Pharmacia Chemical Co., Piscataway, N.J., cellulose and hydroxy apatite resins, distributed by Sigma Chemical Co., St. Louis, Mo., and polystyrene crosslinked with divinylbenzene produced by Dow, Midland, Mi.
The advantages of chromatography are that molecules can be separated on a large scale, relatively rapidly. Disadvantages include loss of materials by non-specific adsorption, uneven packing and particle shifting (void formation and channeling) of the chromatographic material, multiple chromatographies required for complete separation, and large sample sizes.
Electrophoresis is another widely used separation technique in the biologically-related sciences. There are three main ways of using electrophoresis: standard continuous zone electrophoresis, isoelectric focusing, and isotachophoresis. Molecules such as peptides, proteins, and oligonucleotides can be separated by causing them to migrate in a buffer solution under the influence of an electric field. Often the separation medium is a gel such as agarose or polyacrylamide to minimize convective mixing.
In electrophoresis, molecules are primarily separated by different electrophoretic mobilities caused by their different molecular size and/or charge. Other variables affecting separation include the deformabilities and shapes of the macromolecular species. Separations by charge are generally limited to lower molecular weight molecules since the effective charges on a per-mass basis of the larger molecules, such as nucleotides in excess of 1,000 bases, do not differ sufficiently with length. Surfactants such as sodium dodecyl sulphate (SDS), can be used to neutralize the effect of charge differences of most proteins and peptides, so that separation is primarily a function of molecular size, as when proteins are separated by polyacrylamide gel electrophoresis in the presence of SDS (SDS-PAGE). Separations on the basis of charge, irrespective of molecular weight, can also be achieved based on the isoelectric points of the molecules, using a pH gradient, as in isoelectric focusing (IEF).
Early techniques involved imposing an electric field across a slab of gel and placing a sample of material to be analyzed on one end of the gel. Most electrophoretic separations now use commercially available gel columns or slab gel apparatus, distributed, for example, from Pharmacia and Bio-Rad Laboratories. Typically, a monomer solution, one or more crosslinking agents, and surfactants are added to the apparatus and polymerization initiated. An aqueous buffer is usually included to provide an electrically conductive medium in the gel. Apparatus are available both for analytical and for large scale preparative separations.
Recently, microcapillary tubes have been developed for use in microcapillary gel electrophoresis (high-performance capillary electrophoresis, HPCE). These tubes, generally formed of fused silica with an outer polyimide coating, wall thickness in the range of 25 to 40 microns and inner diameter in the range of 25 to 100 microns, are filled with a polyacrylamide gel, the ends placed in an appropriate buffer, and an electric field applied to the gel. The advantage of the HPCE is that the heat resulting from the applied electrical field is efficiently removed due to the high surface area, so a higher current can be applied, thereby decreasing the time required to achieve the desired separation.
All of these methods are still limited as to the separations that can be achieved, particularly of nucleotides and molecules having very similar molecular sizes and charges.
It is therefore an object of the present invention to provide innovative methods and compositions for separation of molecules, particular of molecules that are difficult to separate with existing technology.
It is a further object of the present invention to provide methods and compositions that can be used with existing gel chromatographic and electrophoretic equipment.
It is a still further object of the present invention to provide methods and compositions to separate molecules on both analytical and preparative scales.